Advancing environmental biotechnology with microbial community modeling rooted in functional 'omics.

Emerging biotechnologies that solve pressing environmental and climate emergencies will require harnessing the vast functional diversity of the underlying microbiomes driving such engineered processes. Modeling is a critical aspect of process engineering that informs system design as well as aids diagnostic optimization of performance. 'Conventional' bioprocess models assume homogenous biomass within functional guilds and thus fail to predict emergent properties of diverse microbial physiologies, such as product specificity and community interactions. Yet, recent advances in functional 'omics-based approaches can provide a 'lens' through which we can probe and measure in situ ecophysiologies of environmental microbiomes. Here, we overview microbial community modeling approaches that incorporate functional 'omics data, which we posit can advance our ability to design and control new environmental biotechnologies going forward.

Landfill intermediate cover soil microbiomes and their potential for mitigating greenhouse gas emissions revealed through metagenomics.

Landfills are a major source of anthropogenic methane emissions and have been found to produce nitrous oxide, an even more potent greenhouse gas than methane. Intermediate cover soil (ICS) plays a key role in reducing methane emissions but may also result in nitrous oxide production. To assess the potential for microbial methane oxidation and nitrous oxide production, long sequencing reads were generated from ICS microbiome DNA and reads were functionally annotated for 24 samples across ICS at a large landfill in New York. Further, incubation experiments were performed to assess methane consumption and nitrous oxide production with varying amounts of ammonia supplemented. Methane was readily consumed by microbes in the composite ICS and all incubations with methane produced small amounts of nitrous oxide even when ammonia was not supplemented. Incubations without methane produced significantly less nitrous oxide than those incubated with methane. In incubations with methane added, the observed specific rate of methane consumption was 0.776 +/- 0.055 µg CH4 g dry weight (DW) soil-1 h-1 and the specific rate of nitrous oxide production was 3.64 × 10-5 +/- 1.30 × 10-5 µg N2O g DW soil-1 h-1. The methanotrophs Methylobacter and an unclassified genus within the family Methlyococcaceae were present in the original ICS samples and the incubation samples, and their abundance increased during incubations with methane. Genes encoding particulate methane monooxygenase/ ammonia monooxygenase (pMMO) were much more abundant than genes encoding soluble methane monooxygenase (sMMO) across the landfill ICS. Genes encoding proteins that convert hydroxylamine to nitrous oxide were not highly abundant in the ICS or incubation metagenomes. In total, these results suggest that although ammonia oxidation via methanotrophs may result in low levels of nitrous oxide production, ICS microbial communities have the potential to greatly reduce the overall global warming potential of landfill emissions.

Microbiome assembly and stability during start-up of a full-scale, two-phase anaerobic digester fed cow manure and mixed organic feedstocks.

Carbon transformations during anaerobic digestion are mediated by complex microbiomes, but their assembly is poorly understood, especially in full-scale digesters. Gene-centric metagenomics combining functional and taxonomic classification was performed for an on-farm digester during start-up. Cow manure and organic waste pre-treated in a hydrolysis tank were fed to the methane-producing digester and the volatile solids loading rate was slowly increased from 0 to 3.5 kg volatile solids m-3 d-1 over one year. The microbial community in the anaerobic digester exhibited a high ratio of archaea, which were dominated by hydrogenotrophic methanogens. Bacteria in the anaerobic digester had a high abundance of genes for ferredoxin cycling, H2 generation, and more metabolically complex fermentations than in the hydrolysis tank. In total, the results show that a functionally stable microbiome was achieved quickly during start-up and that the microbiome created in the low-pH hydrolysis tank did not persist in the downstream anaerobic digester.

Diverse electron carriers drive syntrophic interactions in an enriched anaerobic acetate-oxidizing consortium.

In many anoxic environments, syntrophic acetate oxidation (SAO) is a key pathway mediating the conversion of acetate into methane through obligate cross-feeding interactions between SAO bacteria (SAOB) and methanogenic archaea. The SAO pathway is particularly important in engineered environments such as anaerobic digestion (AD) systems operating at thermophilic temperatures and/or with high ammonia. Despite the widespread importance of SAOB to the stability of the AD process, little is known about their in situ physiologies due to typically low biomass yields and resistance to isolation. Here, we performed a long-term (300-day) continuous enrichment of a thermophilic (55 °C) SAO community from a municipal AD system using acetate as the sole carbon source. Over 80% of the enriched bioreactor metagenome belonged to a three-member consortium, including an acetate-oxidizing bacterium affiliated with DTU068 encoding for carbon dioxide, hydrogen, and formate production, along with two methanogenic archaea affiliated with Methanothermobacter_A. Stable isotope probing was coupled with metaproteogenomics to quantify carbon flux into each community member during acetate conversion and inform metabolic reconstruction and genome-scale modeling. This effort revealed that the two Methanothermobacter_A species differed in their preferred electron donors, with one possessing the ability to grow on formate and the other only consuming hydrogen. A thermodynamic analysis suggested that the presence of the formate-consuming methanogen broadened the environmental conditions where ATP production from SAO was favorable. Collectively, these results highlight how flexibility in electron partitioning during SAO likely governs community structure and fitness through thermodynamic-driven mutualism, shedding valuable insights into the metabolic underpinnings of this key functional group within methanogenic ecosystems.

Language trees with sampled ancestors support a hybrid model for the origin of Indo-European languages.

The origins of the Indo-European language family are hotly disputed. Bayesian phylogenetic analyses of core vocabulary have produced conflicting results, with some supporting a farming expansion out of Anatolia ~9000 years before present (yr B.P.), while others support a spread with horse-based pastoralism out of the Pontic-Caspian Steppe ~6000 yr B.P. Here we present an extensive database of Indo-European core vocabulary that eliminates past inconsistencies in cognate coding. Ancestry-enabled phylogenetic analysis of this dataset indicates that few ancient languages are direct ancestors of modern clades and produces a root age of ~8120 yr B.P. for the family. Although this date is not consistent with the Steppe hypothesis, it does not rule out an initial homeland south of the Caucasus, with a subsequent branch northward onto the steppe and then across Europe. We reconcile this hybrid hypothesis with recently published ancient DNA evidence from the steppe and the northern Fertile Crescent.

Comparison of metagenomes from fermentation of various agroindustrial residues suggests a common model of community organization.

The liquid residue resulting from various agroindustrial processes is both rich in organic material and an attractive source to produce a variety of chemicals. Using microbial communities to produce chemicals from these liquid residues is an active area of research, but it is unclear how to deploy microbial communities to produce specific products from the different agroindustrial residues. To address this, we fed anaerobic bioreactors one of several agroindustrial residues (carbohydrate-rich lignocellulosic fermentation conversion residue, xylose, dairy manure hydrolysate, ultra-filtered milk permeate, and thin stillage from a starch bioethanol plant) and inoculated them with a microbial community from an acid-phase digester operated at the wastewater treatment plant in Madison, WI, United States. The bioreactors were monitored over a period of months and sampled to assess microbial community composition and extracellular fermentation products. We obtained metagenome assembled genomes (MAGs) from the microbial communities in each bioreactor and performed comparative genomic analyses to identify common microorganisms, as well as any community members that were unique to each reactor. Collectively, we obtained a dataset of 217 non-redundant MAGs from these bioreactors. This metagenome assembled genome dataset was used to evaluate whether a specific microbial ecology model in which medium chain fatty acids (MCFAs) are simultaneously produced from intermediate products (e.g., lactic acid) and carbohydrates could be applicable to all fermentation systems, regardless of the feedstock. MAGs were classified using a multiclass classification machine learning algorithm into three groups, organisms fermenting the carbohydrates to intermediate products, organisms utilizing the intermediate products to produce MCFAs, and organisms producing MCFAs directly from carbohydrates. This analysis revealed common biological functions among the microbial communities in different bioreactors, and although different microorganisms were enriched depending on the agroindustrial residue tested, the results supported the conclusion that the microbial ecology model tested was appropriate to explain the MCFA production potential from all agricultural residues.

Dwarfism and gigantism drive human-mediated extinctions on islands.

Islands have long been recognized as distinctive evolutionary arenas leading to morphologically divergent species, such as dwarfs and giants. We assessed how body size evolution in island mammals may have exacerbated their vulnerability, as well as how human arrival has contributed to their past and ongoing extinctions, by integrating data on 1231 extant and 350 extinct species from islands and paleo islands worldwide spanning the past 23 million years. We found that the likelihood of extinction and of endangerment are highest in the most extreme island dwarfs and giants. Extinction risk of insular mammals was compounded by the arrival of modern humans, which accelerated extinction rates more than 10-fold, resulting in an almost complete demise of these iconic marvels of island evolution.

Comment on Niemann et al. Outcome Analysis of the Use of Cerament® in Patients with Chronic Osteomyelitis and Corticomedullary Defects. Diagnostics 2022, 12, 1207.

We read the recent study by Niemann et al. (2022) [...].

Mid- to long-term results of single-stage surgery for patients with chronic osteomyelitis using a bioabsorbable gentamicin-loaded ceramic carrier.

AIMS: Excision of chronic osteomyelitic bone creates a dead space which must be managed to avoid early recurrence of infection. Systemic antibiotics cannot penetrate this space in high concentrations, so local treatment has become an attractive adjunct to surgery. The aim of this study was to present the mid- to long-term results of local treatment with gentamicin in a bioabsorbable ceramic carrier. METHODS: A prospective series of 100 patients with Cierny-Mader Types III and IV chronic ostemyelitis, affecting 105 bones, were treated with a single-stage procedure including debridement, deep tissue sampling, local and systemic antibiotics, stabilization, and immediate skin closure. Chronic osteomyelitis was confirmed using strict diagnostic criteria. The mean follow-up was 6.05 years (4.2 to 8.4). RESULTS: At final follow-up, six patients (six bones) had recurrent infection; thus 94% were infection-free. Three infections recurred in the first year, two in the second year, and one 4.5 years postoperatively. Recurrence was not significantly related to the physiological class of the patient (1/20 Class A (5%) vs 5/80 Class B (6.25%); p = 0.833), nor was it significantly related to the aetiology of the infection, the organisms which were cultured or the presence of nonunion before surgery (1/10 with nonunion (10%) vs 5/90 without nonunion (5.6%); p = 0.570). Organisms with intermediate or high-grade resistance to gentamicin were significantly more likely in polymicrobial infections (9/21; 42.8%) compared with monobacterial osteomyelitis (7/79 (8.9%); p < 0.001). However, recurrence was not significantly more frequent when a resistant organism was present (1/16 for resistant cases (6.25%) vs 5/84 in those with a microbiologically sensitive infection (5.95%); p = 0.958). CONCLUSION: We found that a single-stage protocol, including the use of a high-delivery local antibiotic ceramic carrier, was effective over a period of several years. The method can be used in a wide range of patients, including those with significant comorbidities and an infected nonunion.Cite this article: Bone Joint J 2022;104-B(9):1095-1100.

Metagenomes and Metagenome-Assembled Genomes from Microbiomes Metabolizing Thin Stillage from an Ethanol Biorefinery.

Here, we report the metagenomes from five anaerobic bioreactors, operated under different conditions, that were fed carbohydrate-rich thin stillage from a corn starch ethanol plant. The putative functions of the abundant taxa identified here will inform future studies of microbial communities involved in valorizing this and other low-value agroindustrial residues.

Metagenome-Assembled Genomes from a Microbiome Grown in Dairy Manure Hydrolysate.

Anaerobic microbiomes can be used to recover the chemical energy in agroindustrial and municipal wastes as useful products. Here, we report a total of 109 draft metagenome-assembled genomes from a bioreactor-fed carbohydrate-rich dairy manure hydrolysate. Studying these genomes will aid us in deciphering the metabolic networks in anaerobic microbiomes.

Metagenomes and Metagenome-Assembled Genomes from Microbial Communities Fermenting Ultrafiltered Milk Permeate.

Fermentative microbial communities can be utilized for the conversion of various agroindustrial residues into valuable chemicals. Here, we report 34 metagenomes from anaerobic bioreactors fed lactose-rich ultrafiltered milk permeate and 278 metagenome-assembled genomes (MAGs). These MAGs can inform future studies aimed at generating renewable chemicals from dairy and other agroindustrial residues.

Metagenome-Assembled Genomes from a Microbiome Converting Xylose to Medium-Chain Carboxylic Acids.

There is growing interest in producing beneficial products from wastes using microbiomes. We previously performed multiomic analyses of a bioreactor microbiome that converted carbohydrate-rich lignocellulosic residues to medium-chain carboxylic acids. Here, we present draft metagenome-assembled genomes from this microbiome, obtained from reactors in which xylose was the primary carbon source.

Antimicrobial resistance determinants are associated with Staphylococcus aureus bacteraemia and adaptation to the healthcare environment: a bacterial genome-wide association study.

Staphylococcus aureus is a major bacterial pathogen in humans, and a dominant cause of severe bloodstream infections. Globally, antimicrobial resistance (AMR) in S. aureus remains challenging. While human risk factors for infection have been defined, contradictory evidence exists for the role of bacterial genomic variation in S. aureus disease. To investigate the contribution of bacterial lineage and genomic variation to the development of bloodstream infection, we undertook a genome-wide association study comparing bacteria from 1017 individuals with bacteraemia to 984 adults with asymptomatic S. aureus nasal carriage. Within 984 carriage isolates, we also compared healthcare-associated (HA) carriage with community-associated (CA) carriage. All major global lineages were represented in both bacteraemia and carriage, with no evidence for different infection rates. However, kmers tagging trimethoprim resistance-conferring mutation F99Y in dfrB were significantly associated with bacteraemia-vs-carriage (P=10-8.9-10-9.3). Pooling variation within genes, bacteraemia-vs-carriage was associated with the presence of mecA (HMP=10-5.3) as well as the presence of SCCmec (HMP=10-4.4). Among S. aureus carriers, no lineages were associated with HA-vs-CA carriage. However, we found a novel signal of HA-vs-CA carriage in the foldase protein prsA, where kmers representing conserved sequence allele were associated with CA carriage (P=10-7.1-10-19.4), while in gyrA, a ciprofloxacin resistance-conferring mutation, L84S, was associated with HA carriage (P=10-7.2). In an extensive study of S. aureus bacteraemia and nasal carriage in the UK, we found strong evidence that all S. aureus lineages are equally capable of causing bloodstream infection, and of being carried in the healthcare environment. Genomic variation in the foldase protein prsA is a novel genomic marker of healthcare origin in S. aureus but was not associated with bacteraemia. AMR determinants were associated with both bacteraemia and healthcare-associated carriage, suggesting that AMR increases the propensity not only to survive in healthcare environments, but also to cause invasive disease.

Systematic review of risk prediction studies in bone and joint infection: are modifiable prognostic factors useful in predicting recurrence?

Background: Classification systems for orthopaedic infection include patient health status, but there is no consensus about which comorbidities affect prognosis. Modifiable factors including substance use, glycaemic control, malnutrition and obesity may predict post-operative recovery from infection. Aim: This systematic review aimed (1) to critically appraise clinical prediction models for individual prognosis following surgical treatment for orthopaedic infection where an implant is not retained; (2) to understand the usefulness of modifiable prognostic factors for predicting treatment success. Methods: EMBASE and MEDLINE databases were searched for clinical prediction and prognostic studies in adults with orthopaedic infections. Infection recurrence or re-infection after at least 6 months was the primary outcome. The estimated odds ratios for the primary outcome in participants with modifiable prognostic factors were extracted and the direction of the effect reported. Results: Thirty-five retrospective prognostic cohort studies of 92 693 patients were included, of which two reported clinical prediction models. No studies were at low risk of bias, and no externally validated prediction models were identified. Most focused on prosthetic joint infection. A positive association was reported between body mass index and infection recurrence in 19 of 22 studies, similarly in 8 of 14 studies reporting smoking history and 3 of 4 studies reporting alcohol intake. Glycaemic control and malnutrition were rarely considered. Conclusion: Modifiable aspects of patient health appear to predict outcomes after surgery for orthopaedic infection. There is a need to understand which factors may have a causal effect. Development and validation of clinical prediction models that include participant health status will facilitate treatment decisions for orthopaedic infections.

How to Handle Concomitant Asymptomatic Prosthetic Joints During an Episode of Hematogenous Periprosthetic Joint Infection, a Multicenter Analysis.

BACKGROUND: Prosthetic joints are at risk of becoming infected during an episode of bacteremia, especially during Staphylocococcus aureus bacteremia. However, it is unclear how often asymptomatic periprosthetic joint infection (PJI) occurs, and whether additional diagnostics should be considered. METHODS: In this multicenter study, we retrospectively analyzed a cohort of patients with a late acute (hematogenous) PJI between 2005-2015 who had concomitant prosthetic joints in situ. Patients without at least 1 year of follow-up were excluded. RESULTS: We included 91 patients with a hematogenous PJI and 108 concomitant prosthetic joints. The incident PJI was most frequently caused by Staphylococcus aureus (43%), followed by streptococci (26%) and Gram-negative rods (18%). Of 108 concomitant prosthetic joints, 13 were symptomatic, of which 10 were subsequently diagnosed as a second PJI. Of the 95 asymptomatic prosthetic joints, 1 PJI developed during the follow-up period and was classified as a "missed" PJI at the time of bacteremia with S. aureus (1.1%). Infected prosthetic joints were younger than the noninfected ones in 67% of cases, and prosthetic knees were affected more often than prosthetic hips (78%). CONCLUSIONS: During an episode of hematogenous PJI, concomitant asymptomatic prosthetic joints have a very low risk of being infected, and additional diagnostic work-up for these joints is not necessary.

Diagnosing and Predicting Mixed-Culture Fermentations with Unicellular and Guild-Based Metabolic Models.

Multispecies microbial communities determine the fate of materials in the environment and can be harnessed to produce beneficial products from renewable resources. In a recent example, fermentations by microbial communities have produced medium-chain fatty acids (MCFAs). Tools to predict, assess, and improve the performance of these communities, however, are limited. To provide such tools, we constructed two metabolic models of MCFA-producing microbial communities based on available genomic, transcriptomic, and metabolomic data. The first model is a unicellular model (iFermCell215), while the second model (iFermGuilds789) separates fermentation activities into functional guilds. Ethanol and lactate are fermentation products known to serve as substrates for MCFA production, while acetate is another common cometabolite during MCFA production. Simulations with iFermCell215 predict that low molar ratios of acetate to ethanol favor MCFA production, whereas the products of lactate and acetate coutilization are less dependent on the acetate-to-lactate ratio. In simulations of an MCFA-producing community fed a complex organic mixture derived from lignocellulose, iFermGuilds789 predicted that lactate was an extracellular cometabolite that served as a substrate for butyrate (C4) production. Extracellular hexanoic (C6) and octanoic (C8) acids were predicted by iFermGuilds789 to be from community members that directly metabolize sugars. Modeling results provide several hypotheses that can improve our understanding of microbial roles in an MCFA-producing microbiome and inform strategies to increase MCFA production. Further, these models represent novel tools for exploring the role of mixed microbial communities in carbon recycling in the environment, as well as in beneficial reuse of organic residues.IMPORTANCE Microbiomes are vital to human health, agriculture, and protecting the environment. Predicting behavior of self-assembled or synthetic microbiomes, however, remains a challenge. In this work, we used unicellular and guild-based metabolic models to investigate production of medium-chain fatty acids by a mixed microbial community that is fed multiple organic substrates. Modeling results provided insights into metabolic pathways of three medium-chain fatty acid-producing guilds and identified potential strategies to increase production of medium-chain fatty acids. This work demonstrates the role of metabolic models in augmenting multi-omic studies to gain greater insights into microbiome behavior.

Debridement, antibiotics and implant retention (DAIR) for the management of knee prosthetic joint infection.

BACKGROUND: While two-stage revision arthroplasty is viewed as the gold standard for the treatment of knee periprosthetic joint infection (PJI) in terms of infection eradication, it is associated with significant cost along with patient morbidity and mortality. Debridement, antibiotics, and implant retention (DAIR) is an attractive option as it has demonstrated better patient outcomes, comparable implant longevity to primary arthroplasty, and significantly reduced cost when successful. Given the heterogeneity of what is defined as a DAIR the literature is highly variable in terms of its efficacy from the perspective of infection eradication. METHODS: In the setting of a previously well-functioning, well-fixed arthroplasty with an acceptable soft tissue envelope and a treatable organism we report our methods for proceeding with a DAIR procedure, both unicompartmental and total knee. RESULTS: With the above methods we have demonstrated improved patient outcomes when compared to one- or two-stage arthroplasty with lower patient morbidity. Implant longevity in the setting of a successful DAIR is equivalent to those of a primary arthroplasty. CONCLUSIONS: With appropriate indications and good surgical technique as described we believe DAIR is an excellent option in the treatment of periprosthetic joint infection. We hope that with a well-defined protocol as outlined we can gain a better understanding of the efficacy of DAIR procedure with more homogeneity to the procedure to better define when they are most successful while improving patient outcomes and reducing cost.

Lower Success Rate of Débridement and Implant Retention in Late Acute versus Early Acute Periprosthetic Joint Infection Caused by Staphylococcus spp. Results from a Matched Cohort Study.

BACKGROUND: Surgical débridement, antibiotics and implant retention (DAIR) is currently recommended by international guidelines for both early acute (postsurgical) and late acute (hematogenous) periprosthetic joint infections (PJIs). However, due to a different pathogenesis of infection, a different treatment strategy may be needed. QUESTIONS/PURPOSES: (1) Compared with early acute PJIs, are late acute PJIs associated with a higher risk of DAIR failure? (2) When stratified by microorganism, is the higher risk of failure in late acute PJI associated with Staphylocococcus aureus infection? (3) When analyzing patients with S. aureus infection, what factors are independently associated with DAIR failure? METHODS: In this multicenter observational study, early acute and late acute PJIs treated with DAIR were retrospectively evaluated and matched according to treating center, year of diagnosis, and infection-causing microorganism. If multiple matches were available, the early acute PJI diagnosed closest to the late acute PJI was selected. A total of 132 pairs were included. Treatment success was defined as a retained implant during follow-up without the need for antibiotic suppressive therapy. RESULTS: Late acute PJIs had a lower treatment success (46% [60 of 132]) compared with early acute PJIs (76% [100 of 132]), OR 3.9 [95% CI 2.3 to 6.6]; p < 0.001), but the lower treatment success of late acute PJIs was only observed when caused by Staphylococcus spp (S. aureus: 34% versus 75%; p < 0.001; coagulase-negative staphylococci: 46% versus 88%; p = 0.013, respectively). On multivariable analysis, late acute PJI was the only independent factor associated with an unsuccessful DAIR when caused by S. aureus (OR 4.52 [95% CI 1.79 to 11.41]; p < 0.001). CONCLUSIONS: Although DAIR seems to be a successful therapeutic strategy in the management of early acute PJI, its use in late acute PJI should be reconsidered when caused by Staphylococcus spp. Our results advocate the importance of isolating the causative microorganism before surgery. LEVEL OF EVIDENCE: Level III, therapeutic study.